Soft tissue repair allografts and methods for preparing same

ABSTRACT

Allografts for soft tissue repair, including breast reconstruction and other plastic surgery procedures, are disclosed. One allograft is made from decellularized dermal tissue and constitutes a collagen matrix having substantially uniform density and porosity. Another allograft is a hybrid bilayer tissue form that is made from decellularized dermal and adipose tissues. Methods for making both allografts are also disclosed.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional PatentApplication No. 61/783,237, filed Mar. 14, 2013, the disclosure of whichis incorporated in its entirety herein.

FIELD OF THE INVENTION

The present invention relates generally to allografts made fromdecellularized dermal tissues, and in particular, to the use of suchallografts for soft tissue repair, including breast reconstruction andother plastic surgery procedures.

BACKGROUND OF THE INVENTION

Human allograft dermal tissue has been widely accepted for use invarious surgical procedures for decades. For example, acellular dermalmatrices (“ACDMs”) derived from allograft dermal tissue are used in therepair of ventral abdominal hernias and other abdominal wall defects.Commercially available ACDMs include FlexHD® Structural™ ACDM, which ismarketed by Musculoskeletal Transplant Foundation (Edison, N.J.), aswell as AlloDerm® ACDM and AlloDerm® Ready to Use (“RTU”) ACDM, both ofwhich are marketed by LifeCell Corporation (Branchburg, N.J.). Thenature of the dermal tissue from which these ACDMs are derived isexplained with reference to FIG. 1, which illustrates the microstructureof human skin.

Human allograft skin, as illustrated in FIG. 1, is recovered from eitherlive or deceased donors after receiving consent from the individualdonor or donor's family. The skin is made of several layer-likecomponents, including the outer-most epidermis E, and the dermis D,which lies beneath the epidermis. The hypodermis H (also referred to asthe subcutis) lies beneath the dermis D, but is not part of the skin.Rather, the hypodermis H contains adipose and muscle tissue. The dermisD itself includes the papillary dermis PD, which lies adjacent theepidermis E, and the reticular dermis RD, which lies between thepapillary dermis PD and the hypodermis H. The papillary-reticular dermisinterface PRI, lies between the papillary dermis PD and the reticulardermis RD. The dermis-epidermis junction (“the DEJ”) lies between thepapillary dermis PD and epidermis E.

The process for deriving the foregoing ACDMs from dermal tissue involvesremoving the epidermis E (e.g., by a chemical process that causes theepidermis to slough off), and thereby exposing the DEJ that was adjacentthe epidermis E. Beneath the DEJ lies the papillary dermis PD, thepapillary-reticular dermal interface PRI, and the reticular dermis RD.The dermal tissue that is recovered for the ACDMs may therefore includethe DEJ, papillary dermis PD and at least part of the reticular dermisRD. The recovered dermal tissue is decellularized and asepticallyprocessed to meet sterility testing requirements.

The foregoing ACDMs are derived from recovered tissue that includes theentire papillary dermis PD. The microstructure of the papillary dermisPD is not uniform. More particularly, the papillary dermis PD has anupper portion, or side, that was immediately adjacent the DEJ andtherefore closer to the epidermis E (i.e., “the epidermal portion”), anda structurally different lower portion, or side, that was farther fromthe DEJ and epidermis E, and adjacent the deeper reticular dermis RD(i.e., “the dermal portion”). The epidermal portion of the papillarydermis PD contains a more densely-packed collagen matrix than therelatively more open collagen matrix contained in the dermal portion. Assuch, the dermal portion is more porous than the epidermal portion. Thisdual structure is also a property of the foregoing ACDMs, and is idealfor repairing ventral abdominal hernias and other abdominal walldefects, as the more densely-packed epidermal portion of the ACDM (i.e.,incorporating the epidermal portion of the papillary dermis PD)possesses the tensile strength and stiffness required for suchload-bearing tissue repairs, and the more porous dermal portion of theACDM (i.e., incorporating the dermal portion of the papillary dermis PD,as well as at least a portion of the loosely-packed and porousunderlying reticular dermis RD) provides an open collagen structure thatpromotes vascularization, cellular attachment and tissue ingrowth.Nevertheless, this dual structure, which may only be visible on amicroscopic scale, presents concerns about identifying and maintainingthe side orientation of the ACDM, i.e., during a surgical procedure.

Allograft dermal tissue-derived ACDMs have also been used in plasticsurgery procedures, including breast reconstruction, where the ACDM isimplanted to function as an internal sling that is draped around abreast implant and/or tissue expander. While the high tensile strengthand stiffness of the foregoing ACDMs are important for hernia andabdominal wall repairs, breast reconstruction and other plastic surgeryprocedures do not involve the load-bearing and other tissueconsiderations inherent in hernia and abdominal wall repairs. Instead,materials used as slings and similar devices in breast reconstructionshould possess biomechanical properties that are well-suited to suchapplications, including predictable suppleness, flexibility and uniformpliability sufficient for such slings to stretch and expand withouttearing during tissue expansion (i.e., using breast implant and/ortissue expander). Ideal materials for breast reconstruction and otherplastic surgery procedures should also possess sufficient tensilestrength, preclude suture tear-out, both during implantation andexpansion through the post-operative phase, and allow rapid andefficient cellular ingrowth equally from either side of the ACDM.

SUMMARY OF THE INVENTION

The present invention relates to a soft tissue repair allograft, andmore particularly to an allograft dermal tissue-derived ACDM, and itsuse in plastic surgery procedures, including breast reconstruction. TheACDM of the present invention is derived from deeper-cut dermal tissue,which constitutes a collagen matrix having substantially uniform densityand porosity, and therefore possesses the foregoing structural andbiomechanical properties that make it well-suited for use in breastreconstruction procedures, e.g., as a sling, as well as other plasticsurgery applications. The allograft dermal tissue form, or ACDM,includes a portion of dermal tissue having a first exposed surfaceformed by a first cut and a second exposed surface formed by a secondcut opposite the first exposed surface, wherein the portion of dermaltissue constitutes a collagen matrix having substantially uniformdensity and porosity between the first and second exposed surfaces.

The present invention also relates to a method for preparing an ACDM,i.e., an allograft dermal tissue form, from donor tissue. The methodinvolves (1) making a first cut into the reticular dermis RD at a firstlocation distal the papillary-reticular dermis interface PRI, and alonga first plane substantially parallel to the papillary-reticular dermisinterface PRI; (2) removing the hypodermis H from the reticular dermisRD along the first cut to form a first exposed surface on a remainingportion of the donor tissue; (3) making a second cut into the papillarydermis PD at a second location proximate the DEJ, and along a secondplane substantially parallel to the papillary-reticular dermis interfacePRI and the first plane; and (4) removing the epidermis E, DEJ and aportion of the papillary dermis PD from the remaining portion of thedonor tissue to form a second exposed surface on a remaining portion ofthe dermis D opposite the first exposed surface. The first and secondlocations are selected such that the remaining portion of the dermis Dconstitutes a collagen matrix having substantially uniform density andporosity between the first exposed surface and the second exposedsurface.

The present invention further relates to an allograft hybrid bilayertissue form having a dermal side and an adipose side for use in surgicalprocedures, as well as a method for forming the allograft hybrid bilayertissue form. The method involves (1) providing donor tissue includingskin having (a) an epidermis E and (b) a dermis D underlying theepidermis E, the dermis D including a papillary dermis PD adjacent theepidermis E, a reticular dermis RD distal to the epidermis E, and apapillary-reticular dermis interface PRI between the papillary dermis PDand reticular dermis RD; and a hypodermis H adipose tissue underlyingthe reticular dermis RD; (2) making a cut into the reticular dermis RDat a location proximate the hypodermis H, and along a planesubstantially parallel to the papillary-reticular dermis interface PRI;and (3) removing the hypodermis H and a portion of the reticular dermisRD attached to the hypodermis H to form the allograft hybrid bilayertissue form such that the allograft hybrid bilayer tissue form includesboth a dermal side and an adipose side.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further explained with reference to theattached drawings, wherein like structures are referred to by likenumerals and/or letters throughout the several views. The drawings shownare not necessarily to scale, with emphasis instead generally beingplaced upon illustrating the principles of the present invention.

FIG. 1 is a perspective schematic view of a section of human skin andthe various components thereof;

FIG. 2 is perspective schematic view of the section of human skin shownin FIG. 1, and also illustrates the cutting steps performed on sameaccording to an embodiment of the present invention;

FIG. 3 is a cross-sectional schematic view of an ACDM being used as asling for breast reconstruction according to an embodiment of thepresent invention;

FIG. 4 is a perspective view of an ACDM being used as a sling for breastreconstruction according to an embodiment of the present invention;

FIG. 5 a is a perspective schematic view of a prior art process, asperformed on a section of human skin;

FIG. 5 b is a perspective schematic view of a process according to anembodiment of the present invention, as performed on a section of humanskin;

FIG. 6 is a graph of in vitro fibroblast attachment data for variousACDMs;

FIG. 7 is a group of graphs of tensile property data for various ACDMs;

FIGS. 8 a and 8 b are scanning electron micrographs of various ACDMs;

FIGS. 9 a and 9 b are histological images of various ACDMs;

FIG. 10 is a graph of suture retention strength data for various ACDMs;

FIG. 11 a is a plot of standard deviations in the tensile strength datafor various ACDMs;

FIG. 11 b is a plot of standard deviations in the modulus data forvarious ACDMs; and

FIG. 11 c is a plot of standard deviations in the elongation-at-breakdata for various ACDMs.

DETAILED DESCRIPTION OF THE INVENTION

Detailed embodiments of the present invention are disclosed herein. Itshould be understood that the disclosed embodiments are merelyillustrative of the invention that may be embodied in various forms. Inaddition, each of the examples given in connection with the variousembodiments of the invention is intended to be illustrative, and notrestrictive. Further, the figures are not necessarily to scale, and somefeatures may be exaggerated to show details of particular components. Inaddition, any measurements, specifications and the like shown in thefigures are intended to be illustrative, and not restrictive. Therefore,specific structural and functional details disclosed herein are not tobe interpreted as limiting, but merely as examples for teaching oneskilled in the art to variously employ the present invention.

The present invention generally relates to dermal allografts for use inthe repair of soft tissue defects. More particularly, the presentinvention relates to a flexible, pliable acellular dermis surgicalimplant, or tissue form, comprising a section cut from a full thicknessdermal tissue. The ACDMs of the present invention possess structural andbiomechanical properties that are conducive to their use in breastreconstruction and other plastic surgery applications. Such propertiesinclude, but are not limited to, predictable suppleness, flexibility,uniform pliability sufficient to stretch and expand without tearingduring tissue expansion (i.e., using a breast implant and/or tissueexpander), sufficient tensile strength for breast reconstruction andother plastic surgery applications, improved handling properties, andsubstantially uniform porosity that promotes rapid and efficientcellular ingrowth equally from either side of the ACDM.

In one embodiment of the invention, an ACDM is derived from allograftdermal tissue that is recovered from deeper within the dermis, and istherefore farther from, and not adjacent the epidermis. The procedurefor preparing such an ACDM according to one embodiment of the inventionis described below.

The recovery of portions of the dermis D from the skin may beaccomplished by various techniques and devices, such as, for example, amanual dermatome technique, or dissection with a scalpel. In anembodiment illustrated in FIG. 2, a first cut 10 is made into thereticular dermis RD of the skin (e.g., a section of skin cut from theentire donor skin) proximate the underlying hypodermis H in order toremove it from the dermis D. A second cut 20 is then made into theepidermal portion of the papillary dermis PD containing the densecollagen matrix, as discussed in the foregoing Background section, inorder to remove the epidermis E, the DEJ, and the underlying epidermalportion of the papillary dermis PD. The remaining portion of the dermisD (i.e., the deeper dermal portion of the papillary dermis PD and thereticular dermis RD) constitutes a collagen matrix having substantiallyuniform density and porosity.

In one embodiment, the remaining portion of the dermis (“the tissue”) isthen minimally processed, e.g., according to the process disclosed inU.S. Pat. No. 7,723,108, the disclosure of which is incorporated byreference herein in its entirety. In another embodiment, the tissue isdecellularized by chemically treating it with saline, detergent,peracetic acid, ethanol and propylene glycol. The tissue is then washedwith sterile water to remove residual processing chemicals. Thedisinfected and acellular tissue is cut into rectangular-shaped sheetssuitable for clinical uses. The tissue sheets are treated with aqueousethanol and then packaged to provide a hydrated collagen matrix, i.e.,the ACDM.

The process(es) used to treat the tissue preserves the extracellularmatrix of the dermis. The resulting ACDM thereby provides a framework tosupport cellular repopulation, vascularization, and tissue regenerationat the surgical site.

The ACDM derived using the process(es) disclosed above (referred toherein as the “Disclosed ACDM”) exhibits properties that are ideal forits use as a sling in breast reconstruction, and its use in otherplastic surgery applications, as is evident from the Examples presentedbelow. Use of the Disclosed ACDM minimizes adhesions and foreign bodyreactions while promoting vascularization, cellular attachment, andtissue ingrowth at the surgical site. Compared to the prior art ACDMs(i.e., those discussed in the Background section), the Disclosed ACDMpossesses more uniform tensile properties (i.e., strength, pliability,stretchability and handling characteristics) that are optimal for itsuse in breast reconstruction and other plastic surgery applications. TheDisclosed ACDM also possesses improved suture retention strength, andelasticity and deformability that are optimal for its intended use. Forexample, the improved elasticity of the Disclosed ACDM promotes betterexpansion of the tissue in breast reconstruction. The Disclosed ACDM istherefore very strong and closely mimics the biomechanical properties ofthe tissue that it is intended to replace. Further, the Disclosed ACDMis resistant to bacterial colonization and non-immunogenic as a resultof the treatment thereto and decellularization thereof.

FIGS. 3 and 4 illustrate use of the ACDM as a sling for breastreconstruction. As shown in these figures, the ACDM conforms to theshape of the breast implant (or tissue expander) in its function as asupportive sling.

FIG. 5 a illustrates the process for fabricating the prior art ACDMs(i.e., the FlexHD® Structural™ ACDM, AlloDerm® ACDM and AlloDerm® RTUACDM), namely, cutting the lower portion of the dermis and hypodermis(represented by straight line 30), and chemically treating the tissue toremove only the epidermis (represented by uneven line 40) and expose theDEJ.

FIG. 5 b illustrates the process for fabricating the Disclosed ACDMaccording to an embodiment of the present invention. The lower portionof the dermis and hypodermis are cut (represented by straight line 50),and then a second cut (represented by straight line 60), is made deeperinto the dermis than the chemical treatment used to fabricate the priorart ACDMs. In one embodiment, the second cut results in the removal ofthe epidermis, the DEJ, and the upper, epidermal portion of thepapillary dermis.

Presented and discussed below are Examples that illustrate thecomparative biomechanical properties of the Disclosed ACDM and the priorart ACDMs (i.e., the FlexHD® Structural™ ACDM, AlloDerm® ACDM andAlloDerm® RTU ACDM).

Example 1 In Vitro Fibroblast Attachment to the ACDMs Materials andMethods

7 mm punches of each tissue sample (i.e., each ACDM) were prepared andseeded with 1×10⁵ BJ neonatal human foreskin fibroblasts (ATCC, Manassa,Va.) on both sides in Eagles Minimum Essential Medium +10% fetal bovineserum. After 30 minutes, the tissue sections were washed to remove anynon-adherent cells and incubated at 37° C. for 1 hour in complete growthmedium. Attached cells were quantified using CyQuant Cell ProliferationAssay (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. Non-adherent seeded controls were measured for allsamples. The test was replicated with each sample set. Values for cellfluorescence were reported. Tissue from multiple donor lots werecollected, processed as described and tested. In addition, five lots ofAlloDerm® RTU thick tissue were obtained and tested as commercialcontrols.

Results

TABLE 1 In vitro fibroblast attachment No. of Samples Cells* Grouping**FlexHD Structural Dermis 60 6047/242 BC Epidermis 60 2620/270 DDisclosed ACDM Dermis 77 8379/308 A Epidermis 78 7246/359 AB AlloDermDermis 42 4568/476 C Epidermis 42 1548/379 DE AlloDerm RTU Dermis 362028/259 DE Epidermis 36 1039/278 E *Data presented as fluorescenceunits: mean/standard error of the mean, SEM. **Statistically similargroups as determined by the Bonferroni Method (95% Confidence); meansthat do not share a letter are statistically different.

The results presented above are organized to show fibroblast attachmentdata for the dermis side and, separately, the epidermis side of each ofthe ACDMs. These results are similarly organized in the graph of FIG. 6and the following discussion.

Dermal Side of Tissue:

The Disclosed ACDM had a statistically significant higher number ofattached fibroblasts as compared to the FlexHD Structural ACDM; 8379 vs.6047 fluorescence units. The AlloDerm ACDM had a greater number ofattached fibroblasts as compared to the AlloDerm RTU ACDM; 4568 vs.2028. It is noteworthy that the AlloDerm RTU ACDM had less than half asmany attached fibroblasts as compared to the AlloDerm ACDM; this is astatistically significant difference. Finally, the number of attachedfibroblasts for the Disclosed ACDM (8379) was much greater than foreither the AlloDerm ACDM (4568) or AlloDerm RTU ACDM (2028). Thesedifferences are also statistically significant.

Epidermal Side of Tissue:

The Disclosed ACDM had a statistically significant higher number ofattached fibroblasts as compared to the FlexHD Structural ACDM; 7246 vs.2620 fluorescence units. The AlloDerm ACDM had roughly the same level ofattached fibroblasts as the AlloDerm RTU ACDM; 1548 vs. 1039. These weremuch lower than for the FlexHD Structural ACDM or the Disclosed ACDM.Accordingly, the Disclosed ACDM had a much higher level of attachedfibroblasts (7246) as compared to either the AlloDerm ACDM (1548) or theAlloDerm RTU ACDM (1039). The difference between the cell attachmentlevel for the Disclosed ACDM is statistically significantly differentthan for either of the AlloDerm ACDM or the AlloDerm RTU ACDM.

Discussion

The Disclosed ACDM is derived from a deeper cut into the dermis layerrelative to the source of the FlexHD Structural ACDM (see, e.g., FIGS. 5a and 5 b). The porosity of this tissue increases with increased depthinto the dermis. Accordingly, the interconnected channels are larger. Asa corollary, the pores are more uniform at the two surfaces of a deepcut dermis.

In Table 1, the data show that the deeper cut Disclosed ACDM has manymore attached fibroblasts than the FlexHD Structural ACDM. Also, the invitro fibroblast attachment is clearly different for the two sides,dermis and epidermis, of the FlexHD Structural ACDM. For the deeper cutDisclosed ACDM, the in vitro fibroblast attachment is more equal for thetwo sides. Both the AlloDerm and AlloDerm RTU ACDMs have much lowernumbers of attached fibroblasts than do either the Disclosed ACDM or theFlexHD Structural ACDM. The Disclosed ACDM actually has a 76% higherfrequency of fibroblast attachment compared to that of the AlloDerm RTUACDM. The AlloDerm RTU ACDM has a 56% lower frequency of cell attachmentthan that of the AlloDerm ACDM.

Example 2 Tensile Properties of the ACDMs Materials and Methods

Tissue samples (i.e., for each ACDM) were tested on an MTS 858 MiniBionix System. Sample thickness was first measured with a lasermicrometer (Z Mike, Benchmike 4050S). Samples in dogbone configuration(1 cm×7 cm; ASTM 638) were positioned in pneumatic action grips set at29 psi pressure at a gage length of 26 mm. Tissue was pulled to break ata strain rate of 50.6 mm/min. Ultimate tensile strength,elongation-at-break and elastic modulus were recorded. Statisticalanalysis included both tests of the means and the estimates ofvariability for tensile strength, elongation-at-break, and modulus.

Results

As a result of the more open structure and greater porosity of theDisclosed ACDM, as contrasted with the FlexHD Structural ACDM, theDisclosed ACDM has reduced tensile strength as compared to the FlexHDStructural ACDM; 10.97 vs. 15.36 MPa.

As can be seen from the data in Table 2 and the graph illustrated inFIG. 7, the Disclosed ACDM had a tensile strength higher than that ofboth the AlloDerm and AlloDerm RTU ACDMs; (10.97 vs. 9.22 and 9.46 MPa,respectively). These differences are statistically significant.

Modulus is a measure of flexibility. In other words, the greater itsmodulus, the more stiffness a material exhibits. The modulus of theDisclosed ACDM was 38% lower (and therefore less stiff) than that of theFlexHD Structural ACDM; 7.30 vs. 10.14 MPa (see the graph illustrated inFIG. 7). This difference is statistically significant.

The modulus of the Disclosed ACDM is statistically equivalent to that ofthe AlloDerm ACDM; 7.30 vs. 6.98 MPa (see the graph illustrated in FIG.7). The AlloDerm RTU ACDM was, however, less flexible than either theAlloDerm ACDM or the Disclosed ACDM; 8.31 vs. 6.98 or 7.30 MPa. Thesedifferences are statistically significant. Based on the modulus results,the AlloDerm RTU ACDM was 19% stiffer than the AlloDerm ACDM. Thisdifference is statistically significant.

Elongation-at-break is a measure of the amount of stretch before tensilefailure. For this parameter, the Disclosed ACDM and the AlloDerm ACDMwere statistically equivalent; 1.73 vs. 1.62 mm/mm. The AlloDerm RTUACDM, however, had a statistically lower elongation-at-break as comparedto either the Disclosed ACDM or the AlloDerm ACDM; 1.22 mm/mm vs. 1.73or 1.48 mm/mm.

TABLE 2 TENSILE PROPERTIES* DERMAL TISSUES FOR PLASTIC SURGERY ULTIMATEELONGATION-AT- NO. OF NO. OF TENSILE STRENGTH MODULUS BREAK TISSUEDONORS SAMPLES mean/SEM (MPa) Grouping** mean/SEM (MPa) Groupingmean/SEM (%) Grouping Flex HD 5 154 15.36/0.34 A 10.14/0.25  A 1.73/0.04A Structural Disclosed 6 300 10.97/0.21 B 7.30/0.13 C 1.62/0.02 AB ACDMAlloderm 11 88  9.22/0.54 C 6.98/0.38 C 1.48/0.05 B Alloderm 6 100 9.46/0.22 C 8.31/0.22 B 1.22/0.02 C RTU *Data presented asmean/standard error of the mean, SEM. **Statistically similar groups asdetermined by the Bonferroni Method (95% Confidence); means that do notshare a letter are statistically different.

Discussion

Since the porosity of the tissue in the Disclosed ACDM is significantlygreater than that of the FlexHD Structural ACDM, the tensile propertieswere expected to be different; this difference was confirmed. TheModulus, a measure of flexibility, was 38% lower, i.e., more flexiblefor the deeper cut Disclosed ACDM relative to the FlexHD StructuralACDM. Also, the Disclosed ACDM had a higher level of flexibility (13.8%)relative to the AlloDerm RTU ACDM.

The stretchability of these tissues may be expressed in terms of theelongation-at-break data. The stretchability of the Disclosed ACDM andthe AlloDerm ACDM were equivalent. However, the stretchability of theDisclosed ACDM by this measure is 33% higher relative to the AlloDermRTU ACDM.

An expected decrease in tensile strength of 29% was observed in theDisclosed ACDM, relative to that of the FlexHD Structural ACDM. It isnoteworthy that the tensile strength of the Disclosed ACDM was 40%greater than for the AlloDerm ACDM and 39% greater than for the AlloDermRTU ACDM.

Example 3 Surface Characterization of the ACDMs by Scanning ElectronMicroscopy (SEM) Materials and Methods

Tissue samples (i.e., for the Disclosed ACDM and the FlexHD StructuralACDM) were lyophilized and coated with a 10 nm layer of gold. Imageswere taken using a Field Emission Zeiss Scanning Microscope (Carl Zeiss,Inc., Thornwood, N.Y.) with a working distance of 5-10 mm and voltagerange of 30-200 kV. All images were taken at the Department of Ceramicsand Material Science at Rutgers University, New Brunswick, N.J.

Results

Scanning electron micrographs of the epidermal side and the dermal sideof both the FlexHD Structural ACDM and the Disclosed ACDM are presentedin FIGS. 8 a and 8 b, respectively. Representative images were taken at250× for all samples. For both ACDMs, the micrographs of the epidermalside of the ACDMs are shown on the left, and the micrographs of thedermal side are shown on the right.

Discussion

The deeper cut method of the present invention that was used to derivethe Disclosed ACDM results in a different microstructure as compared tothat of the FlexHD Structural ACDM. In contrast to the FlexHD StructuralACDM, the SEM images clearly show the more open and porous structure ofthe Disclosed ACDM. The dermal and epidermal sides are very similar forthe Disclosed ACDM.

Example 4 Surface Appearance of the ACDMs by Histology (Hematoxylin &Eosin Staining) Materials and Methods

Tissue sections (i.e., for the Disclosed ACDM and the FlexHD StructuralACDM) were fixed in 10% neutral buffered formalin prior to paraffinembedding, sectioned and stained via hematoxillin and eosin (H & E). Allhistological processing was performed at Premier Laboratory (Longmont,Colo.). Imaging was also performed at Premier using AperioScope software(Vista, Calif.). Representative images were taken at 10× magnifications.

Results

Images of the stained FlexHD Structural ACDM and the Disclosed ACDM arepresented in FIGS. 9 a and 9 b, respectively. The images are lowmagnification (10×) representative scans of the entire thickness of thetissue samples. In all images, the epidermal side is on the upper partof the scan. However, it should be noted that orientation for thesesamples was not maintained throughout histological processing. In somecases, the samples are virtually symmetrical through the thickness andwhen possible, macrostructural landmarks (such as presence of adipose orhair follicles) were used to identify sidedness.

As expected and illustrated in FIG. 9 a, the FlexHD Structural ACDMshows a dense structure with an even topography on the epidermal side.Towards the dermal side, the structure becomes less dense, with thetissue directly adjacent to the cut edge showing high fragmentation. Onthe other hand, FIG. 9 b shows that the Disclosed ACDM possesses a moreuniform collagen matrix with no distinguishable differences between theepidermal and dermal sides.

Discussion

The histology images are consistent with the SEM images of FIGS. 8 a and8 b, showing the similarity of the dermal and epidermal sides of theDisclosed ACDM. Based on the results in Examples 3 and 4, the DisclosedACDM will cause relatively less confusion and concern about identifyingand maintaining the side orientation thereof, when compared to FlexHDStructural

ACDM and other ACDMs.

Example 5 Suture Retention Strength Testing of the ACDMs Materials andMethods

A size 0 PDS® II suture with a 40 mm, ½ circle tapered needle (Ethicon,Inc., Somerville, N.J.) was placed 5 mm from the edge of 6 cm×1 cm testsamples of the Disclosed ACDM, the FlexHD Structural ACDM and theAlloDerm ACDM. With one end of the sample fixed, the suture was pulledthrough the material of the sample until failure. The load at failurewas recorded on a MTS Mini Bionix System.

Results

TABLE 3 Suture Retention* Suture Retention No. of No. of Strength (MPa)ACDM Sample Donors Samples Mean/SEM Grouping** FlexHD 40 709 3.40/0.03 BStructural ACDM Disclosed ACDM 9 214 4.10/0.07 A AlloDerm ACDM 10 1213.20/0.9  B *Data presented as mean/standard error of the mean, SEM.**Statistically, similar groups as determined by the Bonferroni Method(95% Confidence); means that do not share a letter are statisticallydifferent.

The ability of the Disclosed ACDM to be sutured without tearing (i.e.,its suture retention strength) is statistically significantly higherthan that for the AlloDerm ACDM and the FlexHD Structural ACDM (4.1 vs.3.2 MPa and 4.1 vs. 3.4 MPa, respectively). The suture retentionstrengths of the AlloDerm ACDM and the FlexHD Structural ACDM weresimilar, and equivalent statistically. These results also presented inthe graph of FIG. 10 and further discussed below.

Discussion

The ability of the Disclosed ACDM to resist tearing under load appliedto the suture demonstrates that the Disclosed ACDM has somewhat highersuture pull-out values than that of the FlexHD Structural and AlloDermACDMs.

The higher suture retention strength of the Disclosed ACDM may beattributed to its increased flexibility arising from its more open,porous structure. The resilience provided by this “open net” structurecould account for the higher suture retention strength.

Example 6 Variability of Tensile Properties of the ACDMs Materials andMethods

A comparison of the variability of tensile properties was made betweenthe Disclosed ACDM and the AlloDerm ACDM.

Statistical analyses were made of the standard deviations of the meansfor each tensile parameter: Ultimate tensile strength, Modulus, andElongation-at-break. The standard deviations were compared using twoindependent statistical methods, F-test and Levine's test.

Statistical differences in the variability of the mean is established bytwo independent statistical methods. The standard F-Test demonstrates avery high statistically different level of variability in the tensiledata with a p-value of 0.000. In addition, as a test for data withnon-uniform distribution, the Levine test again demonstrates differencesin the data variability at a statistically significant level with ap-value of 0.016.

Results

The data and results of the statistical analyses are presented in Table4 and FIGS. 11 a, 11 b and 11 c.

For Ultimate Tensile Strength (see FIG. 11 a), the standard deviationfor the Disclosed ACDM (“DP”, left side) was statistically significantlylower than that of the AlloDerm ACDM (“AD”, right side); 3.557 vs.5.076. The statistical difference was valid for both statistical methodsused.

For Modulus (see FIG. 11 b), the standard deviation for the DisclosedACDM (“DP”, left side) was statistically significantly lower than thatof the AlloDerm ACDM (“AD”, right side); 2.260 vs. 3.532. Thestatistical difference was valid for both statistical methods used.

For Elongation-at-break (see FIG. 11 c), the standard deviation of theDisclosed ACDM (“DP”, left side) was statistically significantly lowerthan that of the AlloDerm ACDM (“AD”, right side); 0.33 vs. 0.43. Thestatistical difference was valid utilizing the F-test.

The more uniform tensile properties of the Disclosed ACDM relative tothose of the AlloDerm ACDM can readily be seen in the plots ofindividual values for the three tensile parameters, as shown in FIGS. 11a, 11 b and 11 c.

TABLE 4 VARIABILITY OF TENSILE PROPERTIES Tensile Strength ModulusElongation-at-Break Disclosed Disclosed Disclosed ACDM Alloderm ACDMAlloderm ACDM Alloderm Standard Deviation 3.557 5.076 2.260 3.532 0.3340.434 Sample Size 5/300 11/87 5/300 11/87 5/300 11/88 # Donors/# SamplesStatistically YES YES YES Significant F-Test Levine's Test YES YES  NO**Data for Alloderm Elongation-At-Break is abnormally distributed

Discussion

Variability of the tensile properties is much less for the DisclosedACDM as compared to the Alloderm ACDM. While there appears to be a smalldifference in the actual tensile properties between the Disclosed ACDMand the AlloDerm ACDM there is, however, a very significant differencein the variability of the tensile properties for these two dermalmatrices. For all three tensile properties measured (i.e., tensilestrength, modulus and elongation-to-break), the Disclosed ACDM exhibitsa statistically lower variability of the tensile values than theAlloDerm ACDM. This results in greatly improved uniformity of handlingproperties among individual pieces. Consequently, the Disclosed ACDM isa more predictable tissue form.

To summarize the findings of the above Examples, the process for formingthe Disclosed ACDM minimizes foreign body reactions while promotingvascularization, cellular attachment, and tissue ingrowth. The DisclosedACDM becomes well incorporated into the surrounding tissues whileavoiding adhesions. Tensile properties (strength, pliability andhandling characteristics) of the Disclosed ACDM are optimized. Sutureretention strength and uniformity of tensile properties are alsosignificantly improved for the Disclosed ACDM. The Disclosed ACDM isvery strong and closely mimic the biomechanical properties of the tissuethat it is intended to replace. The Disclose ACDM maintains an optimalelasticity and deformability suited for the intended use, e.g., as asling for use with breast implants and/or tissue expanders in breastreconstruction surgery.

Another allograft tissue form may be simultaneously derived using theprocess disclosed above in connection with the Disclosed ACDM. Moreparticularly, an allograft tissue form is derived by the first cut made10 into the reticular dermis RD of the skin to remove the underlyinghypodermis H, as discussed above and illustrated in FIG. 2. The cutportion of the reticular dermis RD remains attached to the underlyinghypodermis H, and therefore constitutes a “hybrid bilayer” tissue formthat includes both a dermal side and an adipose (i.e., fat) tissue side.Such a tissue form is useful in surgical procedures in which both dermisand adipose tissue are required or desired, as the two tissues may servedifferent functions (e.g., a repair function and a bulking function,respectively). One example of such a surgical procedure is breastreconstructive surgery. Other examples may include various plastic,cosmetic and/or reconstructive surgeries.

It will be understood that the embodiments described herein are merelyexemplary and that a person of ordinary skill in the art may make manyvariations and modifications without departing from the spirit and scopeof the invention. All such variations and modifications are intended tobe included within the scope of the invention, and the appended claims.Some of the possible variations and modifications of the Disclosed ACDMand the dermis/adipose hybrid bilayer tissue form are disclosed below.

The Disclosed ACDM may be provided in particulated form in oneembodiment, depending on the intended surgical use. The dermis/adiposehybrid bilayer tissue form may also be provided in particulated form inone embodiment. In other embodiments, the particulated Disclosed ACDMand/or particulated dermis/adipose bilayer hybrid tissue form may becombined with a carrier, and thereby constitute a flowable tissue form.

In other embodiments, the Disclosed ACDM may be provided in perforatedor meshed form. Perforating the Disclosed ACDM or forming a mesh of theDisclosed ACDM makes it more porous, and ideal for certain surgicalapplications. The dermis/adipose hybrid bilayer tissue form may also beprovided in perforated or meshed form in other embodiments.

In other embodiments, cells may be added to the Disclosed ACDM. Cellsmay also be added to the dermis/adipose hybrid bilayer tissue form. Suchcells may include, for example, stem cells (e.g., embryonic stem cells,mesenchymal stem cells, adult stem cells, skin-derived stem cells, andamnion-derived stem cells), fibroblasts, osteoblasts, myoblasts, andkeratinocytes.

In other embodiments, biological substances may be added to theDisclosed ACDM. Biological substances may also be added to thedermis/adipose hybrid bilayer tissue form. Such biological substancesmay include, for example, platelet-rich plasma (“PRP”), bone marrowaspirate, and/or demineralized bone particles or fibers and/or otherallograft tissue forms. Further, amnion tissue (with or without thenative cells thereof) may be added to the Disclosed ACDM and/or thedermis/adipose hybrid bilayer tissue form, e.g., to function as ananti-adhesion membrane.

In other embodiments, the Disclosed ACDM may be used to wrap around theabove-identified biological substances or other biological substances.In such a wrapper function, the Disclosed ACDM may protect, enclose, andor insulate such biological substances upon implantation. Thedermis/adipose hybrid bilayer tissue form may also be used as a wrapperfor biological substances.

In other embodiments, reinforcing elements may be added to the DisclosedACDM. Reinforcing elements may also be added to the dermis/adiposebilayer tissue form. Examples of such reinforcing elements includeabsorbable fibers and non-absorbable fibers. The reinforcing elementsmay be arranged in various patterns, such as, for example, a gridpattern.

In other embodiments, the Disclosed ACDM may be chemically modified toimbue it with enhanced properties. One example is cross-linking thecollagen of the Disclosed ACDM. The dermis/adipose hybrid bilayer tissueform may also be chemically modified.

We claim:
 1. A method for making an allograft dermal tissue form,comprising the steps of: providing a donor tissue including skin having(a) an epidermis, (b) a dermis underlying the epidermis, the dermisincluding a papillary dermis adjacent the epidermis, a reticular dermisdistal to the epidermis, and a papillary-reticular dermis interface PRIbetween the papillary dermis and reticular dermis, and (c) adermis-epidermis junction between the papillary dermis and epidermis;and a hypodermis adipose tissue underlying the reticular dermis RD,distal to the papillary-reticular dermis interface; making a first cutinto the reticular dermis at a first location distal thepapillary-reticular dermis interface, and along a first planesubstantially parallel to the papillary-reticular dermis interface;removing the hypodermis from the reticular dermis along the first cut toform a first exposed surface on a remaining portion of the donor tissue;making a second cut into the papillary dermis at a second locationproximate the dermis-epidermis junction, and along a second planesubstantially parallel to the papillary-reticular dermis interface andthe first plane; removing the epidermis, dermis-epidermis junction and aportion of the papillary dermis from the remaining portion of the donortissue to form a second exposed surface on a remaining portion of thedermis opposite the first exposed surface, wherein the first and secondlocations are selected such that the remaining portion of the dermisconstitutes a collagen matrix having substantially uniform density andporosity between the first exposed surface and the second exposedsurface.
 2. The method of claim 1, wherein the papillary dermis has anupper portion immediately adjacent the dermis-epidermis junction andproximate the epidermis, and a structurally different lower portiondistal the dermis-epidermis junction and epidermis and adjacent thereticular dermis, wherein the upper portion of the papillary dermis isthe portion of the papillary dermis removed by said second removingstep, and the remaining portion of the dermis includes the lower portionof the papillary dermis and at least a portion of the reticular dermis.3. The method of claim 1, further comprising the step of treating theremaining portion of the dermis.
 4. The method of claim 3, wherein saidtreating step includes decellularizing the remaining portion of thedermis.
 5. The method of claim 3, wherein said treating step includescleaning the remaining portion of the dermis.
 6. The method of claim 3,further comprising the step of cutting the treated remaining portion ofthe dermis into sheets.
 7. The method of claim 6, wherein said cuttingstep is performed so that the sheets have a rectangular shape.
 8. Themethod of claim 3, further comprising the step of packaging the treatedremaining portion of the dermis.
 9. The method of claim 3, furthercomprising the step of adding cells to the treated remaining portion ofthe dermis.
 10. The method of claim 1, further comprising the step ofadding one or more biological substances to the remaining portion of thedermis.
 11. The method of claim 1, further comprising the step of addingone or more reinforcing elements to the remaining portion of the dermis.12. The method of claim 1, further comprising the step of chemicallymodifying the remaining portion of the dermis.
 13. An allograft dermaltissue form for use in surgical procedures, comprising a portion ofdermal tissue having a first exposed surface formed by a first cut and asecond exposed surface formed by a second cut opposite said firstexposed surface, said portion of dermal tissue constituting a collagenmatrix having substantially uniform density and porosity between saidfirst exposed surface and said second exposed surface.
 14. The allograftdermal tissue form of claim 13, wherein said portion of dermal tissueincludes a portion of a papillary dermis and at least a portion of areticular dermis.
 15. The allograft dermal tissue form of claim 14,wherein said portion of said papillary dermis is a lower portion distalan epidermis and adjacent the reticular dermis, said lower portion beingstructurally different from an upper portion of said papillary dermisproximate the epidermis.
 16. The allograft dermal tissue form of claim13, wherein said portion of dermal tissue is in sheet form.
 17. Theallograft dermal tissue form of claim 13, wherein said portion of dermaltissue is in particulate form.
 18. The allograft dermal tissue form ofclaim 13, wherein said portion of dermal tissue is perforated, andthereby in mesh form.
 19. The allograft dermal tissue form of claim 13,further comprising cells.
 20. The allograft dermal tissue form of claim19, wherein said cells include one or more of the group consisting ofstem cells, fibroblasts, osteoblasts, myoblasts and keratinocytes. 21.The allograft dermal tissue form of claim 13, further comprising one ormore biological substances.
 22. The allograft dermal tissue form ofclaim 21, wherein said one or more biological substances are selectedfrom the group consisting of platelet-rich plasma, bone marrow aspirate,demineralized bone particles or fibers other allograft tissue forms, andamnion tissue.
 23. The allograft dermal tissue form of claim 13, furthercomprising reinforcing elements.
 24. The allograft dermal tissue form ofclaim 23, wherein said reinforcing elements are absorbable fibers. 25.The allograft dermal tissue form of claim 23, wherein said reinforcingelements are non-absorbable fibers.
 26. A method for forming anallograft hybrid bilayer tissue form, comprising the steps of: providinga donor tissue including a skin having (a) an epidermis and (b) a dermisunderlying the epidermis, the dermis including a papillary dermisadjacent the epidermis, a reticular dermis distal to the epidermis, anda papillary-reticular dermis interface between the papillary dermis andreticular dermis; and a hypodermis adipose tissue underlying thereticular dermis; making a cut into the reticular dermis at a locationproximate the hypodermis, and along a plane substantially parallel tothe papillary-reticular dermis interface; and removing the hypodermisand a portion of the reticular dermis attached to the hypodermis to formsaid allograft hybrid bilayer tissue form such that said allografthybrid bilayer tissue form includes both a dermal side and an adiposeside.
 27. The method of claim 26, further comprising the step of addingcells to the allograft hybrid bilayer tissue form.
 28. The method ofclaim 26, further comprising the step of adding one or more biologicalsubstances to the allograft hybrid bilayer tissue form.
 29. The methodof claim 26, further comprising the step of adding one or morereinforcing elements to the allograft hybrid bilayer tissue form. 30.The method of claim 26, further comprising the step of chemicallymodifying the allograft hybrid bilayer tissue form.
 31. An allografthybrid bilayer tissue form for use in surgical procedures, comprising adermal side and an adipose side.
 32. The allograft hybrid bilayer tissueform of claim 31, wherein said dermal side includes a portion of areticular dermis RD of a donor skin tissue.
 33. The allograft hybridbilayer tissue form of claim 32, wherein said adipose side includes ahypodermis H underlying the reticular dermis RD of the donor skintissue, and attached to said portion of a reticular dermis RD.
 34. Theallograft hybrid bilayer tissue form of claim 31, wherein said allografthybrid bilayer tissue form is in particulate form.
 35. The allografthybrid bilayer tissue form of claim 31, wherein said allograft hybridbilayer tissue form is perforated, and thereby in mesh form.
 36. Theallograft hybrid bilayer tissue form of claim 31, further comprisingcells.
 37. The allograft hybrid bilayer tissue form of claim 36, whereinsaid cells include one or more of the group consisting of stem cells,fibroblasts, osteoblasts, myoblasts and keratinocytes.
 38. The allografthybrid bilayer tissue form of claim 31, further comprising one or morebiological substances.
 39. The allograft hybrid bilayer tissue form ofclaim 38, wherein said one or more biological substances are selectedfrom the group consisting of platelet-rich plasma, bone marrow aspirate,demineralized bone particles or fibers other allograft tissue forms, andamnion tissue.
 40. The allograft hybrid bilayer tissue form of claim 31,further comprising reinforcing elements.
 41. The allograft hybridbilayer tissue form of claim 40, wherein said reinforcing elements areabsorbable fibers.
 42. The allograft hybrid bilayer tissue form of claim40, wherein said reinforcing elements are non-absorbable fibers.